The role of the exoribonuclease Pacman/Xrn1 in wing development in Drosophila Joseph Alexander Waldron A thesis submitted in partial fulfilment of the requirements of the University of Brighton and the University of Sussex for the degree of Doctor of Philosophy at the Brighton and Sussex Medical School December 2014 Abstract RNA stability plays a critical role in the control of gene regulation by determining the levels of mRNA that can be translated into protein. The aim of this thesis is to investigate the role of the exoribonuclease Pacman in regulating gene expression during wing development in Drosophila melanogaster. Pacman is the only known cytoplasmic exoribonuclease which degrades RNA in a 5’‐3’ direction and is highly conserved across all eukaryotes. The main phenotype of null pacman mutants is that the wing imaginal discs are significantly smaller than wild‐type. The wing imaginal discs are the parts of tissue in the larvae which go on to form the adult wings and are first specified in the embryo. They are an excellent model system to study the growth and development of a multi‐cellular organism as the pathways involved are highly conserved across eukaryotes, including humans. Results from this thesis show that Pacman is required for the correct growth and development of the wing imaginal discs. The reduced size of the wing imaginal discs in pacman mutants was shown to be a direct consequence of the lack of cytoplasmic 5'‐3' exoribonuclease activity within these cells. In addition to the mutant discs being smaller in size they were also delayed in development. Knocking down pacman using RNAi, within specific regions of the disc, also leads to smaller wings and wing vein defects. Using mosaic analysis, it was shown that wing imaginal disc cells have a cell autonomous requirement for Pacman. The cause of the reduced growth and delayed development of pcm14 wing imaginal discs was shown to be an increase in apoptosis. Interestingly, compensatory proliferation was also shown to be occurring in these mutant discs, but clearly this was unable to compensate for the amount of apoptosis occurring. A genetic interaction was observed between pacman and the intrinsic apoptosis pathway in Drosophila. In order to determine whether Pacman was regulating apoptosis directly through this pathway, q‐RT‐PCR was performed, to determine whether any of these pro‐ apoptotic genes were post‐transcriptionally up‐regulated. Results showed that both reaper and hid were significantly up‐regulated, by 8 and 2 fold respectively, in pcm14 wing imaginal disc cells, yet levels of pre‐reaper and pre‐hid were not significantly different to wild‐type. This therefore strongly supports the current hypothesis that Pacman is regulating apoptosis in Drosophila wing imaginal discs by regulating the expression of reaper and hid. Candidates declaration I declare that the research in this thesis, unless otherwise formally indicated within the text, is the original work of the author. The thesis has not been previously submitted to this or any other university for a degree, and does not incorporate any material already submitted for degree. Signed: Dated: 01/12/2014 Acknowledgements I would first of all like to thank my supervisor, Sarah Newbury, both for the opportunity, but most importantly for the unequivocal support throughout my PhD. I am very grateful for her genuine interest and guidance, both personally and professionally. I would also like to thank all members of the lab, both past and present, for all the help and support during my PhD and also for being great friends and colleagues. I would particularly like to thank Dom Grima for the inspiration and supervision during the start of my PhD. Chris Jones deserves a very large thank you for taking over the role of Dom in being my first point of call for any questions/disasters encountered and for the insightful discussions about my work. Maria Zabolotskaya also deserves a big thank you for all the technical skills I have learned and for teaching me the importance of accuracy and tidiness. I would also like to thank Ben Towler for his keen mutual interest both in my work and in football related matters and also to Clare Rizzo‐Singh and Karen Scruby for their often overlooked but never unappreciated support. I would also like to thank Juan Pablo Couso, Claudio Alonso and Robert Ray, in addition to all those who attended the Drosophila retreats over the years, for their critical feedback and enthusiasm. Finally and most importantly, I would like to thank all my family and friends for always being there for me and especially to my partner Lauren. Without her constant love and support this journey would not have been possible. Together we made it! Contents 1 Introduction ........................................................................... 1 1.1 Regulation of gene expression controls the balance of proteins within the cell .... 1 1.1.1 The importance of gene expression ................................................................. 1 1.1.2 The complexities of gene expression ............................................................... 2 1.1.2.1 Transcriptional regulation ............................................................................ 2 1.1.2.2 Post‐transcriptional regulation .................................................................... 4 1.2 The role of mRNA stability in regulating gene expression ....................................... 7 1.2.1 mRNA degradation pathways in eukaryotes ................................................... 7 1.2.1.1 Deadenylation dependent decay ................................................................. 7 1.2.1.2 Deadenylation independent decay ............................................................ 14 1.2.2 P bodies and Stress granules .......................................................................... 16 1.2.3 Quality control mechanisms .......................................................................... 18 1.2.3.1 Nonsense Mediated Decay (NMD) ............................................................ 18 1.2.3.2 Non Stop Decay (NSD) ................................................................................ 19 1.2.3.3 No Go Decay (NGD) .................................................................................... 19 1.2.4 Regulating mRNA half life .............................................................................. 20 1.2.4.1 ARE (AU‐Rich Element) mediated decay .................................................... 20 1.2.4.2 microRNAs .................................................................................................. 22 1.2.5 The importance of RNA stability in development and disease ...................... 26 1.2.5.1 Role of mRNA stability in development ..................................................... 26 1.2.5.2 Diseases associated with RNA stability defects ......................................... 27 1.3 Advantages of studying RNA stability in Drosophila melanogaster ....................... 29 1.3.1 Using Drosophila melanogaster as a model organism ................................... 29 1.3.2 Growth and development of Drosophila melanogaster ................................ 30 1.3.2.1 Life cycle of Drosophila melanogaster ....................................................... 30 1.3.2.2 Hormonal control of development ............................................................ 30 1.3.2.3 Patterning of the embryo ........................................................................... 31 1.3.3 RNA degradation pathways in Drosophila melanogaster .............................. 32 1.4 Using wing imaginal discs to study the regulation of growth and development .. 36 1.4.1 Cell fate determination .................................................................................. 36 1.4.2 Hormonal control of developmental timing .................................................. 37 1.4.3 Regulation of growth by the Hippo pathway ................................................. 39 1.4.4 Regulation of growth through mechanical force ........................................... 40 1.4.5 Cell competition ............................................................................................. 40 1.4.6 Termination of growth ................................................................................... 41 1.5 Background information on the role of Xrn1/Pacman in Drosophila melanogaster development ...................................................................................................................... 42 1.5.1 Structure and conservation of Xrn1 ............................................................... 42 1.5.2 Phenotypes of xrn1 mutants and redundancy between the 5'‐3' and 3'‐5' pathways 45 1.5.3 Expression of Xrn1 during Drosophila development ..................................... 46 1.5.4 Work leading up to this project ..................................................................... 47 1.6 Aims of this project ................................................................................................ 47 2 Materials and Methods ..................................................... 49 2.1 Drosophila stocks ................................................................................................... 49 2.2 Drosophila husbandry ............................................................................................ 52 2.2.1 Drosophila food recipe ................................................................................... 52 2.2.2